Project Abstract Cytosolic calcium overload is a pivotal pathogenic mechanism in Duchenne muscular dystrophy (DMD), a lethal debilitating muscle disease. Elevated cytosolic calcium triggers proteolysis and muscle cell death. Sarco/endoplasmic reticulum calcium ATPase 2a (SERCA2a) is the calcium pump that removes cytosolic calcium in both skeletal and cardiac muscle. Unfortunately, the SERCA2a level is reduced in DMD muscle. Enhancing SERCA2a expression may restore cytosolic calcium homeostasis and reduce muscle disease in DMD. To explore this novel therapy, we generated an adeno-associated viral vector (AAV) to express SERCA2a. We injected the AAV SERCA2a vector via the tail vein to mdx mice, the most commonly used DMD model. Treatment significantly improved skeletal muscle force and heart function. To translate our findings to large mammals, we propose to test AAV SERCA2a therapy in symptomatic DMD dogs, the best large animal model for DMD. We hypothesize that AAV SERCA2a therapy can significantly enhance cytosolic calcium removal and ameliorate skeletal muscle and heart disease in dystrophic dogs. To test this hypothesis, we will pursue two specific aims. In our first aim, we will test whether regional AAV SERCA2a therapy can ameliorate limb muscle disease and improve function. Regional therapy holds potential to improve the life quality of patients, especially these at late-stage. In our second aim, we will test whether systemic AAV SERCA2a therapy can lead to bodywide improvement in affected dogs. DMD affects all muscles in the body. Whole body muscle therapy will result in maximal protection. In summary, our proposed studies will generate the critical large animal data for a future human trial.